Prognostic Factors for Cancer-Specific Survival and Disease-Free Interval of Dogs with Mammary Carcinomas

Canine mammary tumors (CMTs) are the most diagnosed neoplasms in dogs; however, there are few studies analyzing the influence of epidemiological, clinicopathological, and histopathological data on cancer-specific survival (CSS), disease-free interval (DFI), and overall survival (OS) in a large cohort. To contribute to the understanding of the biological behavior of this neoplasm, 385 cases were analyzed, 89% malignant, 4% benign, and 7% non-neoplastic lesions. Among the dogs diagnosed with malignant neoplasms, 86% had early clinical stages (I–III), while 14% had regional or distant metastasis at the time of diagnosis. Carcinoma in a mixed tumor was the most frequent histological type with 44% of the cases and had the best prognosis. Analyzed factors such as the presence of pseudocyesis, previous history of the disease, advanced clinical stage (IV-V), and presence of ulceration obtained significant results for CSS, DFI, and OS through univariate analysis and had a negative impact on the survival of the patients. Multivariate analysis showed that histological grading and age proved to be the best independent parameters for the prognostic evaluation of CSS and DFI in this study. These factors were also significant in the overall survival analysis. Therefore, these parameters should be considered valuable risk and prognostic factors for CMTs.

Te defnitive diagnosis of CMTs is performed through histopathological examination [5,6]. Late diagnosis makes treatment difcult and reduces the survival of afected animals [7,8]. Te primary treatment to control CMTs is surgery [9][10][11][12], which aims to remove the tumor(s) with free margins and prevent the development of new tumors [1]. However, adjuvant therapies can be instituted after surgical treatment [13].
Knowledge of prognostic factors is important to determine therapeutic programs for cancer patients, as it allows the application of diferent therapeutic modalities in an appropriate and individualized way [14,15] which can determine the success of the treatment and maintain the quality of life of cured patients [16]. Tus, the objective of the present study was to analyze risk factors and prognostic criteria through univariate and multivariate analyses of epidemiological data, clinicopathological and histopathological characteristics, and CSS and DFI of female dogs diagnosed with mammary tumors (MTs), to better understand the behavior of this neoplasm and assist in decision making.

Data Collection.
Epidemiological and clinicopathological information of 385 female dogs submitted to mastectomy was obtained by consulting the medical records of the Veterinary Hospital of the Federal University of Viçosa (HVT-UFV), Minas Gerais/Brazil, from January 2012 to December 2020. Te Ethics Committee for the Use of Animals of the Federal University of Viçosa (CEUA/UFV) approved this study and registered it under protocol number 18/2020. Te clinical data collected were as follows: age at diagnosis, body weight, breed, food type, body condition score (BCS), according to Baldwin et al. [17] on cachectic (1), thin (2), normal (3), fat (4), and obese (5), sterilization status, occurrence of pseudocyesis, hormone administration, and history of previous disease (mammary neoplasm, pyometra, or TVT (transmissible venereal tumor)). Te clinicopathological variables analyzed were as follows: tumor location (thoracic, abdominal, and inguinal mammary glands), number of tumors (single or multiple), tumor size (T1: tumors <3 cm; T2: tumors between 3 and 5 cm; and T3: tumors >5 cm), presence of ulceration, regional lymph node status, and presence of distant metastasis.
Te staging and search for metastasis in distant organs were performed by chest radiography in the ventrodorsal and right and left laterolateral views, in addition to abdominal ultrasound evaluation, and routine tests such as blood cell count and biochemical profle were performed at the time of diagnosis of the mammary neoplasm. Te treatment performed on the animals was exclusively surgical resection. Te surgical procedures were classifed as suggested by Fossum [20], as lumpectomy (removal of only the tumor), regional mastectomy (removal of the afected glands and the ones that shared lymphatic drainage, as well as the lymph node associated with the tumor), or radical mastectomy (unilateral removal of all the mammary chain and associated lymph nodes). Te size, location, and number of tumors as well as the clinical status of regional lymph nodes determined the extension of resection.

Histological Processing and Classifcation of the Samples.
Te collected samples were fxed in a 10% bufered formalin solution for 48 hours, dehydrated in increasing solutions of ethyl alcohol, cleared in xylene, and embedded in parafn. Histological sections (3 μm) were obtained, and these sections were stained using the hematoxylin/eosin technique.
Tumors were classifed according to the consensus for the diagnosis, prognosis, and treatment of CMTs [9,12,21]. In addition, the tumors were evaluated according to the Nottingham System classifcation [22]. Te lesion with the worst histological classifcation and consequent worse prognosis was selected in animals with multiple tumors.

Animal Follow-Up.
After surgery, patients were followed up every six months for a minimum period of 12 months, and the animal's owners were contacted by telephone in cases of loss of clinical follow-up. Local progression was categorized as recurrence close to the previous resection site. DFI was defned as the interval between surgery and the development of recurrences and/or nodal and distant metastasis. CSS was defned as the period between surgical excision of the tumor and disease-related death. OS was defned as the period from surgical removal of the tumor until the patient's death from any cause. Animals that were lost to follow-up were not considered for survival analyses.

Statistical Analysis.
Comparisons of categorical variables were performed using the chi-square method. Parametric data were submitted for analysis of variance and Tukey's test, while non-parametric data were submitted to Kruskal-Wallis and Dunn's test, considering the signifcance of P < 0.05 in Prism 5.0 software (GraphPad Inc., CA, USA).
For data analysis, the Statistical Analysis System (SAS OnDemand) was used. Te overall survival time was analyzed by survival analysis (Lifetest Procedure), the comparison among the strata was performed by log-rank and Wilcoxon tests, and the signifcance level adopted was P < 0.05.
Explanatory variables were used to predict the probability of survival for at least 1 year by multivariate logistic regression (Logistic Procedure), backward selection was used, and only variables that were signifcant at P < 0.10 were kept in the fnal model.  Table 2).

Cancer-Specifc Survival (CSS) and Disease-Free Interval (DFI). Animals with non-neoplastic lesions (n = 27)
and benign neoplasms (n = 16) were excluded from survival analyses. Te loss of clinical follow-up and contact with the animal owners occurred in 18.71% (64/342) of the dogs with malignant MTs. Among dogs with malignant MTs, the following were found after complete follow-up: (1) 27.34% (76/278) were alive without recurrence or metastasis, (2) 8.63% (24/278) were alive but with disease progression, (3) 26.98% (75/278) died due to mammary cancer, and (4) 37.05% (103/278) died of unrelated or unknown causes). As a result, 99 dogs (alive with recurrence and dead from the specifc disease) were included in the CSS and DFI analyses, and for OS, 202 dogs were included.
A signifcant association was observed between age, CSS, and DFI. Younger animals (≤9.0 years) had higher median of CSS and DFI (1.460 days, confdence interval 95% IC 95% 730-1.825) and the older ones (>9.0 years) had the lowest (365 days, IC 95% 365-730, P � 0.001; P � 0.001, respectively, Supplementary Figures 1A and 1B; Supplementary Table 2). CSS and DFI did not signifcantly difer according to previous exposure to unknown hormonal contraceptives. CSS probabilities were not signifcantly diferent in animals with or without a history of pseudocyesis; however, animals with a history of pseudocyesis showed an increased DFI in comparison to those never experiencing pseudocyesis (P � 0.034, IC 95% 60-, Supplementary Figure 1C; Supplementary Table 2).
History of previous disease signifcantly infuenced CSS, but not DFI. Dogs with a history of mammary neoplasm had a lower median of CSS (547 days, IC 95% 210-730) and animals without a history of previous disease had a higher median of CSS (730 days, IC 95% 365-1.095, P � 0.021, Supplementary Figure 1D; Supplementary   Table 3. OS was not infuenced by hormone administration, tumor size, ovariohysterectomy, histological classifcation of tumors, and distant metastasis. OS information for the malignant neoplasms studied is detailed in Table 4.

Multivariate Survival Analysis.
Te multivariate Cox regression model included the explanatory variables BCS (score 1-5), histological grade (score I-III), TNM-based clinical stage (initial or advanced), tumor size (score 1-3), pseudocyesis (present or absent), OHE (yes or no), distant metastasis (yes or no), ulceration (present or absent), hormone administration (yes or no), age (years), weight (kg), and number of mammary lesions (n). Te model was able to explain signifcantly the variability seen in the population (P < 0.10). Only the histological grade and age variables remained as independent prognostic factors in the fnal model for CSS and DFI (Table 5; Figure 1).
In CSS, the odds ratio for age was 0.794. Each increase in age by 1 year multiplies the risk of event occurrence (cancerrelated death) by 0.794, and thus each decrease in patient age by 1 year enhances the cancer-specifc survival probabilities by 1.259 times. Te odds ratio for the histological grade is given in Table 6.
In DFI, the odds ratio for age was 0.755. Each increase in age by 1 year multiplies the risk of event occurrence (cancerrelated death) by 0.755, and thus each decrease in patient age by 1 year enhances the disease-free interval probabilities by 1.325 times. Te odds ratio for histological grade is given in Table 6.

Discussion
Epidemiological studies and survival with multivariate analysis are scarce but important to elucidate the biological behavior of CMTs. Tey might be performed using veterinary records that allow the analysis of signifcant amounts of data, defning the prognostic and predictive factors, the characterization of neoplasms, and the observation of tumor progression, which are important for the defnition of CSS and DFI and more appropriate therapy. Tus, the present study is of particular interest due to the cutof of CMTs felt so far, making it a rare report on disease-free interval, specifc survival, and overall survival of CMTs, as most previous studies have focused on the disease-free survival and overall survival only [23][24][25][26][27][28][29][30].
Age is considered a determining factor for the occurrence of MTs, more frequent between 9 and 11 years old [2,19,[31][32][33], with a higher occurrence of malignant neoplasms in older animals [15] and benign neoplasms in young animals [4,32]. In this study, a behavior similar to that described was observed.
Breed is also considered a risk factor for this disease as a result of the existence of a genetic predisposition [2,34,35]. In spite of this, in our study, we did not observe diferences in CSS, DFI, and OS between mixed-breed and purebred animals with MTs, but as expected, purebred animals were more frequent [1,4,6].
Te majority of dogs in this study were not spayed, which could be a risk factor for CSS, DFI, and OS since OHE in early life signifcantly reduces the risk of developing the CMTs [2,34,36,37]. However, the performance or not of OHE in animals with MTs was not signifcant for CSS, DFI, and OS, as observed by Yamagami et al. [23] and Kristiansen et al. [38]. In addition, it was observed that the use of unknown hormonal contraceptive agents did not signifcantly infuence CSS, DFI, and OS, diferent from what was expected, since the prolonged use of contraceptives stimulates the synthesis of growth hormone in the mammary gland [39].
Pseudocyesis is implicated in the pathogenesis of CMTs [40][41][42] and, in this study, proved to be a prognostic factor for DFI and OS. It is still not proven that dogs with a history of malignant MTs are at greater risk of developing new mammary neoplasms [43,44]. However, it was observed that the history of MTs proved to be a prognostic factor for CSS.
Tumors occur more frequently in the caudal abdominal and inguinal mammary glands [45][46][47], probably because in these glands, there is a greater amount of mammary parenchyma and a greater proliferative response to the action Veterinary Medicine International   Veterinary Medicine International of hormones [48], which corroborates the results of this study. Tumor size is considered an independent prognostic factor, and T1 tumors are associated with a better prognosis [12,15,47]. T1 and T2 lesions occurred mainly in carcinoma in a mixed tumor and tubular carcinomas. Animals with nodal and distant metastasis (advanced clinical stage (IV-V)) had lower CSS compared to the initial stage (I-III) of the disease, in agreement with the fndings of Nguyen et al. [15]. Animals with an advanced clinical stage (IV-V) should undergo combined therapy with chemotherapy adjuvant to surgery in an attempt to increase OS [12] and CSS.
In the present study, carcinoma in a mixed tumor was the most frequent histological type, corroborating the fndings of other authors [8,39,49] and presenting a better prognosis and greater CSS, DFI, and OS. Te histological type of the tumor must be taken into account, since histological variations confer diferences in prognosis and treatment [21].  Most MTs in this study were associated with a low histological grade (I-II), resulting in better CSS, DFI, and OS. Tumors with the histological grade III showed a signifcant reduction in CSS, DFI, and OS, as observed by Peña et al. [29] and Nunes et al. [47] in dogs with undiferentiated carcinomas (grade III) that had a worse prognosis than dogs with grade I and II carcinomas. Supporting these data, the application of multivariate logistic regression selected histological grading as an important parameter to assess the clinical outcome. Histological grade was an independent and highly signifcant prognostic parameter for CSS, DFI, and OS.
Ulceration indicates a worse prognosis [9]. Ulceration can be caused by invasive tumor growth or trauma, ischemia, or skin infection, which are characteristics not necessarily associated with aggressive biological behavior [50]. In this study, the presence of tumor ulceration reduced the CSS of the afected animals by 50% and was mainly observed in patients with tumor size 3 and advanced clinical stage (IV-V). Tus, this clinical pathological feature can also be proposed as a prognostic factor for CMTs.
Surgery is the main treatment for MTs [12], except for those with infammatory carcinomas [19,51,52]. CMTs are associated with high rates of morbidity and mortality [9,15,53]; in this study, a mortality rate of 21.63% was observed one year after the diagnosis of the mammary tumor.
We encountered multiple limitations throughout the process of making this research. For this retrospective study to have a larger number of cases analyzed and, therefore, more efective results, we looked over cases from the years 2012 to 2021, checking the hospital's records and manually selecting all of the female dogs that had a mastectomy. However, during this period, the Veterinary Hospital went through several renovations and changed the system, resulting in the loss of a big percentage of the information available from the previous years.
After that, we had to contact the animal owners of the animals we selected, as there was no follow-up described in the medical records. However, some telephone numbers were out of date or had not been provided and some guardians refused to participate in the study, so 60 animals were excluded due to failure to follow up.
Finally, we had all the information we wanted and selected the histological slides of each tumor to have a more recent and adequate histopathological classifcation, according to the consensus and literature suggested in the methodology [12]. Many slides disappeared and had to be redone after fnding the parafn blocks, but some materials were lost due to age or poor processing, excluding these animals from the study. After that, we had a total of 385 dogs with all the necessary factors to enter this retrospective study.
Te only course of treatment used in these animals was the surgical removal of their respective tumors (radical or regional mastectomy or lumpectomy). Te diagnostic method used consisted only of histological classifcation.

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Immunohistochemistry and other techniques are not widely used in our region due to the high cost and difculty in acquiring antibodies. We recognize that the evaluation of biomarkers is extremely important to predict the biological behavior of cancer and to have a better prognosis for each case, but unfortunately, it is not possible to have it in the daily routine of the UFV Veterinary Hospital, which aims to make veterinary medicine accessible to the local population in need while teaching new professionals.

Conclusion
Advanced

Data Availability
Te data used to support the fndings of this study are available on request from the corresponding author.

Ethical Approval
All procedures were performed according to the Ethics Committee for the Use of Animals (CEUA), protocol no. 18/2020.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
ESS, CCR, and CERP conducted laboratory analyses and data interpretation. ESS wrote the article. ESS, CCR, and TBS drafted and revised the article draft. CCR and FLDO translated the paper. APBB, SLMC, and FLV revised the manuscript draft and supervised the laboratory work. ESS and APBB planned and designed the study. All authors have read and approved the fnal version of the article.

Supplementary Materials
Supplementary